Epidemiology and characterization of Providencia stuartii isolated from hospitalized patients in southern Brazil: a possible emerging pathogen

This study aimed to characterize the virulence factors and antimicrobial resistance of Providencia stuartii , an opportunistic pathogen that causes human infections. We examined 45 isolates of P. stuartii both genotypically and phenotypically by studying their adherence to HeLa cells, biofilm formation, cytotoxicity and antimicrobial resistance, and analysed their genomes for putative virulence and resistance genes. This study found that most isolates possessed multiple virulence genes, including fimA, mrkA, fptA, iutA, ireA and hlyA, and were cytotoxic to Vero cells. All the isolates were resistant to amoxicillin plus clavulanic acid, levofloxacin and sulfamethoxazole plus trimethoprim, and most were resistant to ceftriaxone and cefepime. All isolates harboured extended-spectrum beta-lactamase coding genes such as bla CTX-M-2 and 23/45(51.11 %) of them also harboured bla CTX-M-9. The gene KPC-2 (carbapenemase) was detected in 8/45(17.77 %) isolates. This study also found clonality among the isolates, indicating the possible spread of the pathogen among patients at the hospital. These results have significant clinical and epidemiological implications and emphasize the importance of a continued understanding of the virulence and antimicrobial resistance of this pathogen for the prevention and treatment of future infections.


INTRODUCTION
Providencia is a genus composed of ubiquitous facultative anaerobic micro-organisms that are mobile through the use of peritrichous flagella [1].Currently, the genus comprises nine species, two of which, Providencia stuartii and Providencia rettgeri, are most commonly associated with infections in humans [2].
P. stuartii is intrinsically resistant to several commonly used antimicrobials such as first-generation cephalosporins, polymyxins and aminopenicillins.Therefore, treatment is often limited to carbapenems, aminoglycosides and quinolones, and reports of resistance to these agents are increasing frequent [9].
In addition to antimicrobial resistance, pathogens possess other characteristics that enable infection.The ability of pathogenic bacteria to cause diseases in susceptible hosts is facilitated by the combined action of several virulence factors during infection [10].
To date, the prevalence of virulence-related genes in clinical strains of P. stuartii has not been reported, and further studies are needed to determine the prevalent virulence genes in this species.This study aimed to genotypically and phenotypically characterize the virulence factors and antimicrobial resistance of P. stuartii isolates from different human infections.

Bacterial sampling
The 45 P. stuartii strains analysed in this study were isolated from patients hospitalized at the Regional University Hospital of Northern Paraná, Brazil.Isolates were selected for being multidrug-resistant (MDR) according to previously described criteria [11].This classifies multidrug-resistant bacteria as bacteria not susceptible to at least one agent in three or more categories of antimicrobials, using criteria determined by the Clinical Laboratory Standards Institute (CLSI), European Committee for Antimicrobial Susceptibility Testing (EUCAST), or Food and Drug Administration (FDA) of the United States.
The isolates were collected between February 2014 and December 2017.The identification and the antibiogram were determined using the Vitek 2 Compact system (BioMérieux, Marcy-l'Etoile, France).The antibiogram was confirmed using a disc-diffusion test (Kirby-Bauer) according to the CLSI.Samples were tested for amoxicillin plus clavulanic acid, levofloxacin, sulfamethoxazole plus trimethoprim, ceftriaxone, imipenem, cefepime, ertapenem, meropenem, aztreonam and ceftazidime.
This study was approved by the Research Ethics Committee of the State University of Londrina (CEP-1.590.120).This study evaluated 45 bacterial isolates obtained from 34 patients (23 men and 11 women) admitted to our hospital.The patients were aged 24-85 years (mean of 54.67 years).

In silico determination of virulence genes
The sequenced genome of P. stuartii ATCC 33672 (GenBank accession number CP008920) was analysed using the VFanalyzer tool on the Virulence Factor of Pathogenic Bacteria (VFDB) platform (available at http://www.mgc.ac.cn/cgi-bin/VFs/v5/ main.cgi?func=VFanalyzer).The analysis revealed a high sequence identity with previously described virulence genes.The genome of P. stuartii ATCC 33672 was compared to those of Escherichia coli UPEC CFT073 (GenBank NC_004431) and Klebsiella pneumoniae KCTC 2242 (GenBank GCA_000220485.1) strains.The comparison was performed with known enterobacteria, with the aim of verifying possible homologous genes between these pathogens.

Detection of virulence genes
Bacterial DNA was extracted using the boiling method, followed by heat shock.In brief, the isolates were cultured in Luria-Bertani broth for 24 h at 37 °C.Following incubation, the cell culture was transferred to 1.5 ml microtubes and centrifuged at 10 000 r.p.m. for 10 min.After discarding the supernatant, the pellet containing cellular material was resuspended in 300 µl of sterilized ultrapure water.The microtubes were then boiled for 10 min, followed by a 5 min cooling period on ice.Subsequently, the microtubes were centrifuged at 10 000 r.p.m. for 5 min and the resulting bacterial lysate containing the DNA was carefully transferred to another sterile microtube.

HeLa cell-adhesion assay
Cell adhesion was evaluated using a previously described technique [15].The interaction between the cells and bacteria was tested for 6 h.HeLa cells were grown on 13 mm coverslips in 24-well plates containing 1 ml of Dulbecco's modified Eagle's medium (DMEM; Difco).After a cell monolayer was formed, the medium was aspirated and the wells were washed three times with PBS (Na 2 HPO 4 9.7 mM, KH 2 PO 4 1.25 mM, NaCl 137.93 mM, and KCl 2.68 mM).Next, 1 ml of DMEM supplemented with FBS (Difco) and 1 % d-mannose was added to each well, and a 40 µl aliquot of bacterial culture grown in tryptic soy broth (TSB; Difco) was added to each well.The plates were incubated for 3 h at 37 °C, and then the wells were washed three times with 1 ml of sterile PBS to remove non-adherent bacteria.Then, DMEM containing FBS and 1 % d-mannose was added to each well, and the plates were incubated for 3 h at 37 °C.After a 6 h incubation, the wells were washed three times with PBS, and the cells on the coverslips were fixed with 100 % methanol and stained with May−Grünwald stain for 5 min, followed by Giemsa stain for 20 min.The coverslips were observed under a light microscope at 1000×magnification.HeLa cells were used as the negative controls.As positive controls, we used E. coli strain E2348/69 for localized adhesion [16], E. coli C1845 for diffuse adhesion [17] and E. coli 042 for aggregative-type adhesion [18].

Biofilm formation assay
All isolates were subjected to a biofilm formation assay performed in polystyrene 96-well plates using crystal violet as described previously [19].E. coli 042 [18] was used as the positive control, and TSB (Difco) was used as the negative control.Absorbance (A) was measured at 570 nm using a spectrophotometer, and biofilm formation was classified as absent, weak, moderate, strong or very strong [19].

Haemolytic activity assay
Haemolytic activity on blood agar containing 5 % sheep blood was evaluated using a previously described method [20].To prepare the blood agar plates, freshly sterilized and cooled nutrient agar medium (45-50 °C) was aseptically supplemented with 5 % sheep blood.The test isolates were then inoculated on the blood agar plates, which were incubated overnight at 37 °C.After incubation, the haemolytic activity of the isolates was examined, and the isolates that produced a clear zone were considered haemolytic.

Assay for cytotoxicity against Vero cells
The cytotoxicity of all isolates was assessed against Vero cells (African green monkey kidney) as described previously [21].All isolates were cultivated in 3 ml of TSB (Difco) for 18 h at 37 °C with shaking at 180 r.p.m.The broth was then centrifuged at 13 000 g for 10 min.The supernatants were collected and filtered (47 mm PVDF syringe filters with 0.22 µm pores; Durapore).Filtered supernatants were serially diluted (1 : 10) in 96-well polystyrene plates and incubated for 72 h at 37 °C and 5 % CO 2 .
Cytotoxicity of the isolates was quantified by measuring the metabolic activity of Vero cells incubated with the bacteria supernatant using the (3-[4,5-dimethylthiazol-2-yl]−2,5-diphenyltetrazolium) MTT assay [22].The supernatant of E. coli O157:H7 (EDL933) was used as a positive control for cytotoxicity [23].Vero cells without the bacterial culture supernatant were used as negative controls.A strain was considered highly cytotoxic when cell death was ≥50 % compared to the negative control.
Each 25 µl reaction contained 2 mM MgCl 2 (Invitrogen), 10×buffer (Invitrogen), 1.25 mM dNTPs (Invitrogen), 50 pmol of forward primer, 50 pmol of reverse primer (Invitrogen), 2 U of Taq DNA polymerase (Invitrogen) and 100 ng of DNA template.For DNA amplification, the cycling conditions consisted of an initial denaturation step of 5 min at 94 °C, followed by 40 cycles of denaturation for 1 min at 94 °C, primer annealing for 1 min at 50 °C, and extension for 5 min at 72 °C, and a final extension step at 72 °C for 10 min.The PCR products were subjected to 1.5 % agarose gel electrophoresis for 4 h in TBE buffer, stained with ethidium bromide (0.5 µg ml −1 ), and visualized using a UV transilluminator (Vilbert Loumart).
A similarity dendrogram was constructed using the Gel J 2.0 software [25], unweighted pair group method with arithmetic mean (UPGMA), and data similarity coefficient for cluster analysis (Jaccard).The standard cutoff for defining a cluster was 85 %, which was the standard cutoff for Gram-negative bacteria used for epidemiological purposes in a previous study [26].

Virulence genotypes of the isolates
Genome analysis of P. stuartii ATCC 33672 revealed several putative genes with high sequence identity to those encoding common virulence factors in other enterobacteria (Tables S1 and S2, available in the online version of this article).Six of these were selected for analysis of the clinical isolates: two fimbriae genes (fimA and mrkA), three siderophore receptors (ireA, fptA and iutA), and haemolysin (hlyA) (Fig. S1).All isolates analysed had virulence-related genes that may contribute to their pathogenesis in humans.The fimA, mrkA, fptA, iutA and hlyA genes were present in all analysed isolates, and the ireA gene was found in 93.33 % of isolates.

Virulence phenotypes of the isolates
Phenotypic characterization showed that all isolates adhered to HeLa cells without d-mannose supplementation.However, in the presence of d-mannose supplementation, only 18/45 isolates (40 %) adhered to HeLa cells, whereas the remaining 27/45(60 %) did not (Fig. 1).
All the isolates formed biofilms.However, the extent of biofilm formation varied.Among the isolates, (10/45) 22.22 %, (22/45) 48.89 % and (13/45) 28.89 % were strong, moderate and weak biofilm formers, respectively (Fig. S2).The cytotoxicity against Vero cells revealed that all isolates were cytotoxic to Vero cells, and 33/45 isolates (73.33 %) were considered highly cytotoxic, as they killed 50 % or more of the cultured cells.Haemolytic activity was investigated on blood agar, and all isolates were found to be haemolytic, showing lysis zones on blood agar after 24 h of incubation (Fig. S3).

Genetic similarity among the isolates
The examined isolates showed 85 % similarity, and nearly all isolates, except isolates 10 and 14, grouped into one large cluster (clonal complex A).Three sub-clusters were formed with >90 % similarity (Fig. 2): sub-cluster A1 consisted of 23 isolates, subcluster A2 consisted of eight isolates, and sub-cluster A3 consisted of 11 isolates.One isolate, 45, did not fit into any sub-cluster.There were differences in the phenotypes, virulence and resistance genes among the isolates in each sub-cluster, In sub-cluster A1, there was a predominance of micro-organisms isolated in 2016 (Table 2), and most isolates were positive only for bla CTX-M-2 , although three isolates also harboured bla CTX-M-9 .All isolates had the same virulence genes, and phenotypic characteristics varied among the isolates.
In sub-cluster A2, only two isolates lacked the bla CTX-M-9 gene.Two isolates lacked ireA, which encodes a siderophore receptor.
The phenotypic characteristics varied among the isolates.In sub-cluster A3, there was a predominance of micro-organisms isolated in 2014, and all isolates harboured the beta-lactamaseencoding genes bla CTX-M-2 and bla CTX-M-9 , as well as all the virulence-related genes studied, and their phenotypic characteristics were similar.
Several isolates were derived from the same patient: isolates 2 and 6 were from patient 2; isolates 13 and 15 were from patient 10; isolates 17 and 43 were from patient 12; isolates 24 and 27 were from patient 18; isolates 35, 37 and 39 were from patient 28; and isolates 40 and 41 were from patient 31 (Table 2).However, these strains were isolated on different dates or from different origins, indicating that these patients had multiple infections with clonal isolates of P. stuartii.Interestingly, several isolates from the same patient, that is, isolates 10 and 11 (patient 8), isolates 12 and 14 (patient 9), and isolates 4, 8 and 23 (patient 4), were not grouped in the same sub-clusters despite being from the same patient, indicating that these patients were infected with different micro-organisms.

DISCUSSION
This study aimed to identify virulence-related genes in clinical isolates of P. stuartii and to associate them with their pathogenicity phenotypes.Comparison of the genome of P. stuartii ATCC 33672 with those of other enterobacteria revealed genes in P. stuartii with high identity to virulence-related genes common to these micro-organisms.Of the identified genes, six were chosen for analysis of the studied isolates: fimbriae-(fimA and mrkA), siderophore receptor-(ireA, pftA, and iutA) and hemolysin-(hlyA) encoding genes.
The fimA gene encodes the largest subunit of type 1 fimbriae, which are involved in host-cell adhesion and colonization [27].All the 45 isolates tested positive for fimA.In a study by Daga et al. [28], the prevalence of type 1 fimbria-encoding genes in analysed E. coli isolates was 95.8 %.In another study of E. coli isolates by Kakian et al. [29], the frequency of fimA was 74 % in isolates from hospital inpatients and 76 % in isolates from outpatients.In a study by Ghasemian et al. [30], the prevalence of fimA in Klebsiella oxytoca was 60 %.These data indicate a high prevalence of type 1 fimbria-encoding genes in enterobacteria.
The mrkA gene encodes a subunit of type 3 fimbriae.These fimbriae mediate the attachment to host epithelial cells and play important roles in biofilm formation on abiotic surfaces [31].Ghasemian et al. [30] showed the mrkA gene was present in 42 % of K. oxytoca isolates.
Iron is an essential element for bacterial growth.Bacteria acquire iron by secreting siderophores that bind to iron present in their hosts.Invasive E. coli strains have high-affinity iron acquisition systems that compete with the siderophores of the host and favour bacterial growth when the iron content is low.IreA, iutA and fptA encode receptors that bind iron-chelating siderophores [32].In the present study, iutA and fptA were detected in all isolates, whereas ireA was detected in 93.33 % of the analysed micro-organisms.
In their study on P. mirabilis, Sanches et al. [33] detected ireA in all studied isolates, demonstrating its high prevalence, similar to that observed in the present study.In a study by Daga et al. [28], iutA was observed in 64.3 % of E. coli isolates.Kadry et al. [34] detected the pyochelin siderophore receptor-encoding gene fptA in 85.7 % of evaluated Pseudomonas aeruginosa isolates, indicating a high prevalence of this gene and corroborating the findings of the present study.
The hlyA gene encodes a haemolysin that lyses red blood cells.Despite being known for its haemolytic function, it also has cytotoxic activity against a wide range of cell types and is therefore considered an important virulence factor [35].
Type 1 fimbriae, such as those encoded by the fim operon, are commonly found in enterobacteria such as E. coli and are involved in the attachment of bacterial cells to the surface of specific receptors on eukaryotic cells, thus enabling adhesion [36].Type 1 fimbriae contribute to the increased virulence of E. coli in the urinary tract, thereby promoting bacterial persistence [37].The expression of type 1 fimbriae in the cellular envelope of uropathogenic E. coli allows attachment to the epithelium of the bladder and lower urinary tract, leading to colonization and the establishment of infection [38].Type 1 fimbriae are also considered important virulence factors in K. pneumoniae, as they mediate the initial adhesion to host cells [39].In this study, we analysed the isolates for the presence of two genes encoding the largest subunits of type 1 and 3 fimbriae: fimA and mrkA.In adhesion tests, type 1 fimbriae are inhibited by d-mannose; however, under these conditions, adhesion using other adhesins, such as type 3 fimbriae, can occur.Only 18 of the analysed isolates adhered to HeLa cells in an aggregative manner in the presence of d-mannose, whereas all isolates adhered without d-mannose.This could be explained by the inhibition of type 1 fimbriae by d-mannose.
Biofilm formation is vital for bacterial colonization at the start of an infection, whether or not the infection is associated with a device [40].Mobley et al. [41] reported that P. stuartii isolates expressing MR/K fimbriae adhere to catheters more easily than isolates that do not express this type of adhesin.MR/K fimbriae are related to the persistence of a pathogen in the catheter as, after adhesion, a biofilm can form, which contributes to the establishment of an infection.The expression of type 3 fimbriae is essential for biofilm formation in various bacterial species [39].
Type 3 fimbriae may not be directly involved in urinary tract infections (UTIs) but may be essential in device-associated infections such as catheter-associated UTI.Thus, type 3 fimbriae-encoding genes such as mrkA play an essential role in biofilm formation in K. pneumoniae, which is important for initial cell-to-surface fixation and cell-cell adhesion [39].
E. coli haemolysin, encoded by the hlyA gene, is one of the main virulence factors and the best-studied haemolysin.HlyA is a member of a large family of exotoxins produced by several Gram-negative bacteria, including Proteus and Morganella spp.
Haemolysin lyses erythrocytes via osmotic shock, resulting in hydrophilic pores on the cell surface [42].E. coli haemolysin produces a clear zone around colonies on blood agar owing to erythrocyte lysis [43].The ruptured erythrocytes release haemoglobin, and haemoglobin from lysed erythrocytes is an excellent source of iron because it contains four haem groups, each bound to an iron molecule released through cell lysis [44].
After haemoglobin is released from erythrocytes, bacterial cells must take up haem.Several pathogenic bacteria secrete molecules called siderophores, such as those encoded by the genes examined in this study, which bind to ferrous iron with high affinity and transport it back to the bacteria, or encode receptors that bind haem directly [44,45].
Cytotoxicity analysis revealed that all the tested isolates showed some cytotoxicity against Vero cells, and 33/45 isolates were considered highly cytotoxic, killing 50 % or more of the cultured cells.Many factors such as the production of toxins and proteases can influence the cytotoxicity of pathogens.For example, although HlyA is cytolytic towards erythrocytes, it has also been reported to lyse other cell types in a variety of human and animal hosts [46].
At high concentrations, HlyA forms multimeric pores in eukaryotic membranes, leading to cell lysis, whereas at lower concentrations, it can interfere with host-cell signalling pathways and cause cell death by apoptosis [45].HlyA from E. coli induces oxidative stress, leading to the accumulation of free radicals and cell death due to oxidative damage [47].
In the present study, all isolates had phenotypic characteristics of ESBL producers, and the investigation of β-lactamase genes by PCR revealed that all 45 isolates carried the bla CTX-M-2 gene and 23 isolates carried the bla CTX-M-9 gene, corroborating the study by Liu et al. [48], where all P. stuartii isolates tested were considered ESBL.
In a study by Zavascki et al. [49] carried out at a Brazilian university hospital, all analysed P. stuartii isolates were also positive for bla CTX-M-2 ; however, no other bla CTX-M genes were found.The bla CTX-M-2 , bla CTX-M-8 and bla CTX-M-9 genes are most commonly found in ESBL-producing enterobacteria in South American countries [50].In Brazil, bla CTX-M-2 is one of the most prevalent genes, along with bla CTX-M-15 , which occurs more frequently in the southeastern region [51].
Since the first description of bla KPC genes, carbapenemase-producing K. pneumoniae isolates have become a major concern worldwide.Other enterobacteria carrying the bla KPC-2 gene have also been reported; however, P. stuartii isolates carrying this gene are uncommon.Isolates of P. stuartii carrying the bla KPC-2 gene have already been described by Tavares et al. [52] and Aires et al. [53], and the isolates carrying this gene were found in our study.This clearly demonstrates the need for the immediate recognition of these isolates, so that control measures can be established to prevent their spread within hospitals.
Based on the results presented in the dendrogram (Fig. 2), it is possible to state that there is similarity between the isolates analysed.However, we cannot conclude whether these isolates are clonally related or are the result of intrahospital transmission.Therefore, we suggest that future studies be conducted to elucidate this issue.
Some studies have reported clonal relationships among P. stuartii strains of clinical origin.In a study by Saida et al. [54], two clones of P. stuartii were shown to be disseminated among patients in a burn ward, and in a study by Douka et al. [55], a pan-resistant clonal strain of P. stuartii was shown to be the cause of infections in an intensive care unit.These studies indicate the possibility of the nosocomial dissemination of P. stuartii.
These pathogens can originate from the patient's own microbiota and can be transferred to the site of infection.In addition, pathogens can be disseminated by the treatment team through direct contact with patients.Treatment teams can also transport pathogens to other environments and other patients [56].
Another alarming factor in outbreaks caused by P. stuartii is the resistance of these micro-organisms to commonly used antimicrobials, in addition to the resistance genes acquired by horizontal gene transfer.This highlights the clinical importance of P. stuartii as an emerging pathogen and the concerns regarding its ability to acquire and disseminate resistance genes [9].

CONCLUSION
Based on our findings, it can be concluded that the P. stuartii isolates exhibit several virulence traits that make them highly successful pathogens.These include a strong capacity for adhesion to HeLa cells, cytotoxicity to Vero cells, haemolytic activity on blood agar, and the ability to form biofilms.Additionally, all isolates contained genes encoding various virulence factors, such as fimbriae, haemolysin, and iron and haem acquisition systems.In particular, all tested isolates were positive for ESBL and genes encoding beta-lactamases, indicating a high potential for antimicrobial resistance.This was further supported by the fact that all the isolates were resistant to many commonly used antimicrobials, with [(8/45) 17.77 %] isolates carrying the bla KPC-2 gene.These findings emphasize the need for ongoing efforts to prevent the spread of drug-resistant strains of P. stuartii in hospitals.Clonality was revealed among the isolates through ERIC-PCR typing, suggesting that a single isolate may be responsible for multiple infections in hospitalized patients.This highlights the importance of implementing effective infection control measures, promoting proper hygiene practices and continually monitoring the emergence of new strains of P. stuartii to prevent the nosocomial transmission of these pathogens.-Author response to reviewer 1 in relation to how similarity was determined between isolates was sufficient, but the explicit description of how similarity was determined was not added into the actual manuscript text.Please add this description to the manuscript for reader clarity.

Peer review history
Answer:Thank you very much for the suggestions.Corrections have been made.New information has been added in section "2.9 Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR)".Lines 190-194 -In the absence of sequencing data to support this claim, the statements in lines 385-389 are not appropriate to include in the manuscript.Please remove these statements or provide additional data that shows evidence of within-hospital transmission.
Answer:Thank you very much for the suggestions.We made the suggested corrections.We agree that we cannot say that there is intra-hospital transmission.Therefore, we withdraw these statements and suggest future studies.Line 377 -379 -Minor English grammar issues are still present throughout the text.Please carefully revise the manuscript as a whole to remove these errors.
Answer:We appreciate your feedback and have taken it into account.The revised version of the manuscript has undergone English language editing by Editage.We would like to confirm that only the English grammar has been revised, and no changes to the scientific content have been made.

VERSION 2
Editor  Comments to Author: Guidone and colleagues present an analysis of clinical Providencia stuartii isolates including for the presence of antibiotic resistance genes and virulence factors, and cytotoxicity, adhesion and biofilm formation.They also examine the presence of VFs in the genome of a model P. stuartii genome.
Overall, I think their work is interesting and is suited to Access Microbiology.I think it has relevance to clinical microbiologists.I do however have several major concerns that will need to be addressed: 1.The Results is lacking raw data in the form of tables or figures.It is okay to use the text to summarise the data, but I would like to see it all presented so that it can be analysed independently by the reader.I have included some specific suggestions for each section below.
Answer to Reviewer 1:We agree.Corrections performed.New data have been added as supplemental figures in the results section.
2. The authors refer repeatedly to the clinical isolates being 'resistance to X/Y/Z antibiotics' or having 'resistance phenotypes'.However, to the best of my knowledge the authors have not conducted any antibiotic susceptibility testing in vitro, and therefore cannot make these claims.They instead need to refer to the data they are presenting, i.e. the presence of resistance genes found using PCR with specific primers.
Answer to Reviewer 1:We agree.Corrections performed.It really wasn't clear in the text that phenotypic tests for resistance were also done.This information has been added to the text in section 2.1 Bacterial sampling, lines 84 -87.
3. The authors appear to suggest in the Abstract and Discussion that they have evidence of within-hospital transmission.I am not happy that they are showing this with the data presented.I would need to see whole genome sequencing as a minimum, with e.g.analysis of single nucleotide polymorphisms, to be able to make this claim.
Answer to Reviewer 1:We agree with reviewer suggestion that utilizing whole genome sequencing would indeed be the ideal approach to definitively confirm within-hospital transmission.However, we would like to clarify that the technique we employed, ERIC-PCR, can provide valuable evidence of transmission within the hospital setting.While we acknowledge that whole genome sequencing, including the analysis of single nucleotide polymorphisms, would provide a higher level of resolution, we believe that our study's findings, as presented through ERIC-PCR, still contribute to the understanding of potential transmission dynamics.Furthermore, we'd like to highlight the practical constraints we encountered.Due to the substantial number of isolates in our study and the associated technical and financial limitations, conducting whole genome sequencing for all strains was not feasible within our current resources.Therefore, we chose ERIC-PCR as a practical alternative that could offer meaningful insights into transmission patterns.In conclusion, while we concur that whole genome sequencing would offer a more comprehensive perspective, we believe that the evidence from our ERIC-PCR approach is indicative of within-hospital transmission.We appreciate your consideration of these constraints and our rationale for the chosen methodology.
Minor comments and further details below: Abstract: Would benefit from a little more detail e.g.reference the CTX-M genes as extended-spectrum beta-lactamases, KPC-2 as a carbapenemase to help readers who might not be familiar with gene names.
CTX-M genes referred to as CTM-M several times.
Answer to Reviewer 1:We agree.Corrections performed.

Methods:
Line 71-73: Please provide a brief overview of the criteria used to categorise the isolates as MDR, alongside reference 11.
Section 2.1: Do the authors have genome sequences for the clinical isolates?Are they short/long read, 16S?Please provide details and ensure that the sequences are publicly available, if appropriate.
Answer to Reviewer 1:We don't have the sequences of 16S.The isolates were identified using the Vitek® 2 Compact system.Lines 82-84.
Line 86 -87: How related is P. stuartii to E. coli and K. pneumoniae i.e. how relevant are these as comparisons?
Answer to Reviewer 1:Our aim was to compare the genome of P. stuartii with that of well-studied and established enterobacteria.This comparison aimed to determine the presence of shared virulence genes among these bacterial species.Given the lack of prior descriptions of virulence-related genes in P. stuartii, our intention was to shed light on the pathogenic potential of this species.
Line 90-91: Provide more details of the DNA extraction protocol so that it could be replicated easily.
Answer to Reviewer 1:Corrections performed.The protocol was better described.Lines 105-113. Results: Line 183: Please list e.g. in a supplementary table the putative virulence factors in ATCC 33672 and their similarity to known VFs.Is this the first time this particular species has been interrogated for the presence of VFs?This could be made clearer.
Answer to Reviewer 1:The putative genes found in the comparison between the genomes were added to Supplementary Tables 1 and 2.
Section 3.1: It is not clear from the text whether the authors have looked for VFs in silico or in vitro.Please provide more details in text and an accompanying figure e.g.table of putative VFs or gel images.
Answer to Reviewer 1:A photograph of the PCR product gel for the researched genes has been included in the supplementary material.Supplementary Figure 1.
Line 198: The legend for Figure 1 is not clear -'Adhesion of P. stuartii in HeLa cells' is repeated unnecessarily and is confusing.
It would be useful to include the treatment in the legend e.g. the addition of D-mannose supplementation to the cells in panel B.
Answer to Reviewer 1:The suggested corrections were performed.
Line 200-202: Please provide a figure or table to support the biofilm results (could be Supplementary) e.g. the raw data.Percentages on their own are not adequate without an idea of numbers tested.
Answer to Reviewer 1:A figure containing the biofilm data has been added to the supplementary material as Supplementary Figure 2.
Line 205-206: Please provide example images showing the hemolysis or other examples of the raw data.
Answer to Reviewer 1:The figure demonstrating the hemolysis formed by P. stuartiiisolates has been added to the supplemental material as Supplementary Figure 3.
Section 3.3 (line 208): As far as I can tell, the authors have not conducted in vitro antibiotic susceptibility testing.This section describes isolates as being 'resistant to amoxi/clav…etc' (lines 209-212).If the authors are inferring this from the presence of antibiotic resistance genes then this should be made clear, else details of MIC testing should be provided in Methods.
Answer to Reviewer 1:Text corrections have been made.Antimicrobial tests were performed using the Vitek 2 system.However, knowing the limitations of the method, all antimicrobial susceptibility tests were performed using the disk-diffusion method (as described in section 2.1 Bacterial Sampling) to confirm the results.
Line 208: The section title is misleading -from what I can tell there is no phenotype information being given, only the presence of resistance genes.A more appropriate section header could be something like 'Presence of resistance genes in clinical isolates' (or something more informative, if the authors choose).
Answer to Reviewer 1:We appreciate your observation regarding the section title and agree that it could potentially lead to confusion.While the phenotypic antibiotic testing was detailed in section 2.1, we understand that the current title might not accurately reflect the content.To address this concern, we have revised the section title to "Antibiotic resistance of clinical isolates".This title better encapsulates the information presented in the section and clarifies that both phenotypic antibiotic testing and the presence of resistance genes are discussed.
Figure 2: The legend is not as informative as it could be -for example, are the six VFs mentioned in the table the only ones found by bioinformatic analysis, or are they a subset chosen by the authors?The cytotoxicity (against what target) should be mentioned.Please also made the +/-signs a little larger for ease of reading.Why is the adhesion data in the presence of D-mannose not provided here?
Answer to Reviewer 1:The genes related to virulence factors analyzed were a subset chosen by the authors among all that were found.The genes were chosen because they are common genes for other enterobacteria.To elucidate this selection, the text has been revised accordingly (Lines 207-211).Additionally, the text now specifies the target cell for cytotoxicity.The recommended alterations in Figure 2 have been incorporated, encompassing the outcomes in the presence of D-mannose.
Line 218: How has this measure of similarity been determined?How were the clonal complexes determined?
Answer to Reviewer 1:The similarity was determined by grouping isolates with similar band patterns, performed by UPGMA and Jaccard´s coefficient, using the GelJ program.The 85% cut-off was established for studies of genetic similarity of enterobacteria in previous study (reference 26).Upon establishing the defined threshold (indicated by the red line in Figure 2), the clusters that emerged exhibiting a similarity exceeding this level constituted what we refer to as "clonal complexes" in this study.
Line 217: I believe this should be section 3.4, rather than 3.3 Answer to Reviewer 1:Correction performed.
Line 218-235: "The phenotypic characteristics varied/were similar' -which characteristics are these?There is a lot of information being presented here so please be clear Answer to Reviewer 1:The phenotypic characteristics identified as variables in this context were the phenotypic traits, such as biofilm formation and cytotoxicity, which typically exhibit variations when assessed across different isolates.The manuscript has been revised accordingly, with the modifications made in lines 224 to 241.
Line 218-235: Is there a characteristic common to all isolates in a given sub-cluster?From Fig 2 there seems to be a lot of intracluster variability, so I am intrigued to understand how they were clustered in this way.
Answer to Reviewer 1:The ERIC analysis amplifies repetitive regions prevalent in enterobacteria.These amplified regions yield distinct fingerprint patterns that facilitate the classification of similar isolates based on these amplified regions.Notably, while the isolates displayed substantial variability in phenotypic characteristics, the majority of the examined isolates exhibited the presence of identical virulence genes. Discussion: In contrast to the Introduction, the Discussion is lacking structure and focus.It reads more like an informative passage on VFs rather than a discussion of the results presented in the manuscript.I strongly encourage the authors to consider a re-write to focus on what their results mean in a clinical context.
Line 342: What phenotypic characteristics of ESBL producers are the authors referring to?
Answer to Reviewer 1:The characteristics that were being referred to is resistance to beta-lactam antimicrobials.The text has been revised for better clarity.Line 373.
Line 363-356: This is a bold claim, and would benefit from more information being provided to support it e.g. which antimicrobials were being used that could have acted as a selection pressure.
Answer to Reviewer 1:In this context, we deliberate on a potential array of microorganisms based on their phenotypic resistance and the presence of resistance genes.Regrettably, we lack access to information regarding the specific antimicrobials employed during the patient's treatment.
Line 366-374: I am not clear whether the authors are merely repeating claims from other papers, or whether they are suggesting that they have also found evidence of within-hospital transmission of P. stuartii.If the latter, I am not convinced that they are able to state this from the data provided.I would expect to see whole genome sequences and SNP analyses to be able to make such a claim.
Answer to Reviewer 1:The statements presented in the discussion provide an overview of other studies that have identified clonal relationships among the examined isolates.In our study, we aimed to address this aspect as well.However, we acknowledge that based on the conducted tests, we cannot definitively assert that the isolates are exact clones of each other, consequently only implying a clonal relationship between them.

Reviewer 2
Comments to Author: The study "Epidemiology and characterization of KPC-2-producing Providencia stuartii isolated from hospitalized patients in southern Brazil: A possible emerging pathogen" describes that P. stuartii presents a high frequency of virulence genes commonly found in other Enterobacterales species.The authors demonstrated that increased adhesion capacity to HeLa cells, citotoxicity in Vero cells, hemolytic activity and can form biofilm.

Major comments:
The experiments were well designed and are well described, although some minor questions could be completed.
The results were well presented.
In the discusion section, the authors could improve by trying to correlate the genes found with the results of the adhesion, citotoxicity and biofilm tests.
A minor english revision is suggested.

Minor comments:
The title indicates that only KPC-2 isolates would be assessed regarding epidemiology and characterization, while all isolates were actually evaluated.
"CTM-M" in the abstract should be corrected to "CTX-M".
Answer to Reviewer 2:We agree.The title has been corrected.Corrections were made throughout the text.
In Results section, it is suggested to report the absolute number, for example "33/45 (73.33%)".When refering to genes, the nomeclature should be corrected, such as blaCTX-M (with bla in italics and CTX-M underscript), blaKPC-2 etc.If suitabible, it would be interesting to add another columm to figure 2 with the patient.In the Conclusion section, the percentage of KPC-2 positive isolates is different from the one reported in the abstract and in the Results section.

Please rate the manuscript for methodological rigour Good
Please Comments: Guidone and colleagues present an analysis of clinical Providencia stuartii isolates including for the presence of antibiotic resistance genes and virulence factors, and cytotoxicity, adhesion and biofilm formation.They also examine the presence of VFs in the genome of a model P. stuartii genome.Overall, I think their work is interesting and is suited to Access Microbiology.I think it has relevance to clinical microbiologists.I do however have several major concerns that will need to be addressed: 1.
The Results is lacking raw data in the form of tables or figures.It is okay to use the text to summarise the data, but I would like to see it all presented so that it can be analysed independently by the reader.I have included some specific suggestions for each section below.2. The authors refer repeatedly to the clinical isolates being 'resistance to X/Y/Z antibiotics' or having 'resistance phenotypes'.However, to the best of my knowledge the authors have not conducted any antibiotic susceptibility testing in vitro, and therefore cannot make these claims.They instead need to refer to the data they are presenting, i.e. the presence of resistance genes found using PCR with specific primers.3. The authors appear to suggest in the Abstract and Discussion that they have evidence of within-hospital transmission.I am not happy that they are showing this with the data presented.I would need to see whole genome sequencing as a minimum, with e.g.analysis of single nucleotide polymorphisms, to be able to make this claim.
Minor comments and further details below: Abstract: Would benefit from a little more detail e.g.reference the CTX-M genes as extended-spectrum beta-lactamases, KPC-2 as a carbapenemase to help readers who might not be familiar with gene names.CTX-M genes referred to as CTM-M several times.Introduction is concise and covers all relevant information.Methods: Line 71-73: Please provide a brief overview of the criteria used to categorise the isolates as MDR, alongside reference 11.Section 2.1: Do the authors have genome sequences for the clinical isolates?Are they short/long read, 16S?Please provide details and ensure that the sequences are publicly available, if appropriate.Line 86 -87: How related is P. stuartii to E. coli and K. pneumoniae i.e. how relevant are these as comparisons?Line 90-91: Provide more details of the DNA extraction protocol so that it could be replicated easily.Results: Line 183: Please list e.g. in a supplementary table the putative virulence factors in ATCC 33672 and their similarity to known VFs.Is this the first time this particular species has been interrogated for the presence of VFs?This could be made clearer.Section 3.1: It is not clear from the text whether the authors have looked for VFs in silico or in vitro.Please provide more details in text and an accompanying figure e.g.table of putative VFs or gel images.Line 198: The legend for Figure 1 is not clear -'Adhesion of P. stuartii in HeLa cells' is repeated unnecessarily and is confusing.It would be useful to include the

Fig. 1 .
Fig. 1.Adhesion of P. stuartii in HeLa cells.(a) Adhesion of P. stuartii in HeLa cells.(b) Bacterial cells not adhered by inhibition of type 1 fimbriae by dmannose treatment.

2 Londrina
https://doi.org/10.1099/acmi.0.000652.v3.1 © 2023 Tolman L. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.Lindsey Tolman; University at Albany, UNITED STATES Date report received: 08 October 2023 Recommendation: Accept Comments: All editor and reviewer comments have been sufficiently addressed and we welcome your manuscript for publication in Access Microbiology.Author response to reviewers to Version and characterization of Providencia stuartiiisolated from hospitalized patients in southern Brazil: A possible emerging pathogenWe thank reviewers/editor for their helpful suggestions, which allowed us to significantly improve the edition of our manuscript, as well as the expression of the ideas.All modifications in the text are highlighted in yellow. of Providencia stuartiiisolated from hospitalized patients in southern Brazil: A possible emerging pathogen

Author response to reviewers to Version 1 Londrina
and characterization of KPC-2-producing Providencia stuartii isolated from hospitalized patients in southern Brazil: A possible emerging pathogenWe thank reviewers for their helpful suggestions, which allowed us to significantly improve the edition of our manuscript, as well as the expression of the ideas.All modifications in the text are highlighted in yellow.

Table 1 .
Oligonucleotide primers of virulence-related genes of P. stuartii.

Table 2 .
Origin of the studied P. stuartii isolates

Table 2 .
Continued While most criticisms have been sufficiently addressed, some minor issues raised by reviewers persist in the revised manuscript.Please be sure to completely correct for and address the following: -Author response to reviewer 1 in relation to how similarity was determined between isolates was sufficient, but the explicit description of how similarity was determined was not added into the actual manuscript text.Please add this description to the manuscript for reader clarity.
-In the absence of sequencing data to support this claim, the statements in lines 385-389 are not appropriate to include in the manuscript.Please remove this section or provide additional data that shows evidence of within-hospital transmission.-Minor English grammar issues are still present throughout the text.Please carefully revise the manuscript as a whole to remove these errors.If additional help is needed, we offer a discounted translation service, Editage (https://www.editage.com/; see https://www.microbiologyresearch. org/ prepare-an-article# 13 for more information).
rate the quality of the presentation and structure of the manuscript GoodTo what extent are the conclusions supported by the data?Strongly supportDo you have any concerns of possible image manipulation, plagiarism or any other unethical practices?NoIs there a potential financial or other conflict of interest between yourself and the author(s)?NoIf this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?YesReviewer 1 recommendation and comments https://doi.org/10.1099/acmi.0.000652.v1.3 © 2023 Anonymous.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.